Methods and reagents for the enhancement of virus transduction in the bladder epithelium

ABSTRACT

Agents and methods for enhancing recombinant virus transduction in the bladder epithelium are described. A first method involves contacting the luminal surface of the bladder with a composition comprising a transduction enhancing agent and an oncolytic virus. Alternatively, the luminal surface of the bladder can be contacted first with a pretreatment composition comprising a transduction enhancing agent and, subsequently, with a composition comprising an oncolytic virus. Bladder treatment compositions comprising a transduction enhancing agent and an oncolytic virus are also described.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to the treatment ofbladder cancer with viral therapy agents and, in particular, to agentsand methods for enhancing recombinant oncolytic virus transduction ofthe bladder epithelium.

[0003] 2. Background of the Technology

[0004] Bladder cancer is a commonly occurring cancer and more than50,000 new cases are diagnosed every year. Bladder cancer is asuperficial disease confined to the mucosa in the majority of patients.Of the various therapeutic modalities available, transurethralresectioning of the tumor is considered to be the most effectivetreatment for the management of superficial bladder cancer. However, 70%of these superficial bladder tumors will recur after endoscopicresectioning, and 20% progress to life-threatening invasive diseaseswithin 2 years of cystectomy. See Raghavan et al., “Biology andManagement of Bladder Cancer”, N. Engl. J. Med., 322, 16, 1129-1138(1990).

[0005] Gene therapy has also been used for the treatment of bladdercancer. See, for example, Brewster et al., Eur. Urol. 25, 177-182(1984); Takahashi, et al., Proc. Natl. Acad. Sci. USA 88, 5257-5261(1991); and Rosenberg, J. Clin. Oncol., 10, 180-199 (1992).

[0006] In vitro studies using cell lines derived from human bladdertissues have demonstrated efficient transgene expression followinginfection with recombinant adenovirus. Bass, et al., Cancer Gene Therapy2, 2, 97-104 (1995). Experiments in vivo have also shown adenovirustransgene expression in the urinary bladder of rodents afterintravesical administration. Bass, et al., supra; Morris, et al., J.Urology, 152, 506-550 (1994). In vitro experiments with wild-typeadenovirus demonstrate that virus attachment and internalization is notinfluenced by benzyl alcohol, but do demonstrate an enhanced uncoatingof the virion. Blixt, et al., Arch. Virol., 129, 265-277 (1993).

[0007] In vivo studies have demonstrated that various agents (e.g.acetone, DMSO, protamine sulfate) can break down the protective “mucin”layer that protects the bladder epithelium from bacteria, viruses andother pathogens. See, for example, Monson et al., J. Urol., 145, 842-845(1992) and Parsons, et al., J. Urol., 143, 139-142 (1990). Methods ofmodifying the bladder surface to enhance gene transfer have also beendisclosed. Siemens, et al., “Evaluation of Gene Transfer Efficiency byViral Vectors to Murine Bladder Epithelium”, J. of Urology, 165, 667-671(2001).

[0008] U.S. Pat. No. 6,165,779 discloses a gene delivery systemformulated in a buffer comprising a delivery-enhancing agent such asethanol or a detergent. The gene delivery system may be a recombinantviral vector such as an adenoviral vector.

[0009] There still exists a need, however, for improved gene therapymethods and agents which can accomplish direct, optimal, in vivo genedelivery to the bladder epithelium.

SUMMARY OF THE INVENTION

[0010] According to a first aspect of the invention, a method fortreating cancer of the bladder is provided. According to this aspect ofthe invention, the method involves: contacting the luminal surface ofthe bladder with a pretreatment composition comprising a transductionenhancing agent; and subsequently contacting the luminal surface of thebladder with a composition comprising an oncolytic virus; wherein thetransduction enhancing agent is a mono-, di-, or poly-saccharide havinga lipophilic substituent. The transduction enhancing agent can have thefollowing general formula (I) or the following general formula (II):

[0011] wherein X is a sulfur or oxygen atom, R¹ is an alkyl group andeach R² is independently hydrogen or a moiety represented by:

[0012] wherein R¹ is an alkyl group. The pretreatment composition canfurther include an oxidizing agent. The oncolytic virus can be anoncolytic adenovirus such as CG8840. The oncolytic virus composition canfurther include a chemotherapeutic agent such as docetaxel.

[0013] According to a second aspect of the invention, a method fortreating cancer of the bladder is provided. According to this aspect ofthe invention, the method includes contacting the luminal surface of thebladder with a pretreatment composition comprising about 0.01 to about0.2% by weight sodium oxychlorosene and, subsequently, contacting theluminal surface of the bladder with a composition comprising anoncolytic virus.

[0014] According to a third aspect of the invention, a method oftreating cancer of the bladder is provided. According to this aspect ofthe invention, the method includes: contacting the luminal surface ofthe bladder with a pretreatment composition comprising a transductionenhancing agent having a structure represented by the chemical formula:

[0015] wherein x and y are positive integers; and subsequentlycontacting the luminal surface of the bladder with a compositioncomprising an oncolytic virus. According to a preferred embodiment ofthe invention, x is 6 and y is 8-10 and the pretreatment compositioncomprises about 0.02 to about 0.05 wt. % of the transduction enhancingagent.

[0016] According to a fourth aspect of the invention, a method oftreating cancer of the bladder is provided. According to this aspect ofthe invention, the method includes: contacting the luminal surface ofthe bladder with a pretreatment composition comprising a transductionenhancing agent having a structure represented by the following generalformula (I) or the following general formula (II):

[0017] wherein x is a positive integer and subsequently contacting theluminal surface of the bladder with a composition comprising anoncolytic virus.

[0018] According to a fifth aspect of the invention, a compositioncomprising a transduction enhancing agent and an oncolytic virus isprovided. According to this aspect of the invention, the transductionenhancing agent is a mono-, di-, or poly-saccharide having a lipophilicsubstituent. For example, the transduction enhancing agent can be acompound having the following general formula (I) or the followinggeneral formula (II):

[0019] wherein X is a sulfur or oxygen atom, R¹ is an alkyl group andeach R² is independently hydrogen or a moiety represented by:

[0020] wherein R¹ is an alkyl group. The oncolytic virus can be anoncolytic adenovirus such as CG8840. The oncolytic virus composition canfurther include a chemotherapeutic agent such as docetaxel. A method fortreating cancer of the bladder comprising contacting the luminal surfaceof the bladder with a composition as set forth above is also provided.

[0021] According to a sixth aspect of the invention, a compositioncomprising sodium oxychlorosene and an oncolytic virus is provided. Theoncolytic virus can be an oncolytic adenovirus such as CG8840. Theoncolytic virus composition can further include a chemotherapeutic agentsuch as docetaxel. A method for treating cancer of the bladdercomprising contacting the luminal surface of the bladder with acomposition as set forth above is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention may be better understood with reference tothe accompanying drawings in which:

[0023]FIGS. 1A and 1B are photographs showing a murine bladder afterpretreatment with a 15% ethanol solution followed by infection withAd-LacZ wherein FIG. 1A shows the outside surface of the bladder andFIG. 1B shows the luminal bladder surface;

[0024]FIGS. 1C and 1D are photographs showing a murine bladder afterpretreatment with a 20% ethanol solution followed by infection withAd-LacZ wherein FIG. 1C shows the outside surface of the bladder andFIG. 1D shows the luminal bladder surface;

[0025]FIGS. 1E and 1F are photographs showing a murine bladder afterpretreatment with a 25% ethanol solution followed by infection withAd-LacZ wherein FIG. 1E shows the outside surface of the bladder andFIG. 1F shows the luminal bladder surface;

[0026]FIGS. 1G and 1H are photographs showing a murine bladder afterpretreatment with a 30% ethanol solution followed by infection withAd-LacZ wherein FIG. 1G shows the outside surface of the bladder andFIG. 1H shows the luminal bladder surface;

[0027]FIG. 2A ia a photograph showing a cross section of a murinebladder control;

[0028]FIGS. 2B and 2C are photographs showing the cross section of amurine bladder after pretreatment with a 30% ethanol solution followedby infection with Ad-LacZ;

[0029] FIGS. 3A-3F are photographs showing the cross section of a murinebladder after pretreatment with a 25% ethanol solution followed byinfection with Ad-LacZ wherein FIGS. 3A, 3C and 3E were taken at 40× andFIGS. 3B, 3D and 3F were taken at 100× magnification;

[0030] FIGS. 4A-4F are photographs showing the cross section of a murinebladder after pretreatment with a 30% ethanol solution followed byinfection with Ad-LacZ wherein FIGS. 4A, 4C and 4E were taken at 40× andFIGS. 4B, 4D and 4F were taken at 100× magnification;

[0031] FIGS. 5A-5D are photographs showing two murine bladders afterpretreatment with a 4% poloxomer 407 solution followed by infection withAd-LacZ wherein FIGS. 5A and 5B show the outside and luminal surfaces,respectively, of the first bladder and FIGS. 5C and 5D show the outsideand luminal surfaces, respectively, of the second bladder;

[0032] FIGS. 6A-6D are photographs showing two murine bladders afterinfection with a composition comprising lipofectamine and Ad-LacZwherein FIGS. 6A and 6B show the outside and luminal surfaces,respectively, of the first bladder and FIGS. 6C and 6D show the outsideand luminal surfaces, respectively, of the second bladder;

[0033] FIGS. 7A-7D are photographs showing two murine bladders afterinfection with a composition comprising In vivo geneSHUTTLE™ and Ad-LacZwherein FIGS. 7A and 7B show the outside and luminal surfaces,respectively, of the first bladder and FIGS. 7C and 7D show the outsideand luminal surfaces, respectively, of the second bladder;

[0034] FIGS. 8A-8N are photographs showing seven murine bladders afterpretreatment with a 0.2% oxychlorosene solution for 5 minutes followedby infection with Ad-LacZ wherein FIGS. 8A and 8B show the outside andluminal surfaces, respectively, of the first bladder, FIGS. 8C and 8Dshow the outside and luminal surfaces, respectively, of the secondbladder, FIGS. 8E and 8F show the outside and luminal surfaces,respectively, of the third bladder, FIGS. 8G and 8H show the outside andluminal surfaces, respectively, of the fourth bladder, FIGS. 81 and 8Jshow the outside and luminal surfaces, respectively, of the fifthbladder, FIGS. 8K and 8L show the outside and luminal surfaces,respectively, of the sixth bladder, and FIGS. 8M and 8N show the outsideand luminal surfaces, respectively, of the seventh bladder;

[0035] FIGS. 9A-9N are photographs showing seven murine bladders afterpretreatment with a 0.2% oxychlorosene solution for 15 minutes followedby infection with Ad-LacZ wherein FIGS. 9A and 9B show the outside andluminal surfaces, respectively, of the first bladder, FIGS. 9C and 9Dshow the outside and luminal surfaces, respectively, of the secondbladder, FIGS. 9E and 9F show the outside and luminal surfaces,respectively, of the third bladder, FIGS. 9G and 9H show the outside andluminal surfaces, respectively, of the fourth bladder, FIGS. 91 and 9Jshow the outside and luminal surfaces, respectively, of the fifthbladder, FIGS. 9K and 9L show the outside and luminal surfaces,respectively, of the sixth bladder, and FIGS. 9M and 9N show the outsideand luminal surfaces, respectively, of the seventh bladder;

[0036]FIGS. 10A and 10B are photographs showing the cross section of themurine bladders of FIGS. 8C and 8I, respectively;

[0037]FIGS. 11A and 11B are photographs showing the cross section of themurine bladders of FIGS. 9C and 9I, respectively;

[0038] FIGS. 12A-12F are photographs showing the cross section of amurine bladder after pretreatment with a 0.2% oxychlorosene solution for5 minutes followed by infection with Ad-LacZ wherein FIGS. 12A, 12C and12E were taken at 40× and FIGS. 12B, 12D and 12F were taken at 100×magnification;

[0039] FIGS. 13A-13F are photographs showing the cross section of amurine bladder after pretreatment with a 0.2% oxychlorosene solution for15 minutes followed by infection with Ad-LacZ wherein FIGS. 13A, 13C and13E were taken at 40× and FIGS. 13B, 13D and 13F were taken at 100×magnification;

[0040]FIG. 14A is a photograph showing the luminal surface of a murinebladder after pretreatment with a 0.1% oxychlorosene solution followedby infection with Ad-LacZ;

[0041]FIGS. 14B and 14C are photographs showing the cross section of themurine bladder of FIG. 14A wherein FIG. 14B was taken at 40× and FIG.14C was taken at 100× magnification;

[0042]FIG. 15A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.2% oxychlorosene solutionfollowed by infection with Ad-LacZ;

[0043]FIGS. 15B and 15C are photographs showing the cross section of themurine bladder of FIG. 15A wherein FIG. 15B was taken at 40× and FIG.15C was taken at 100× magnification;

[0044]FIG. 16A is a photograph showing the luminal surface of a secondmurine bladder after pretreatment with a 0.2% oxychlorosene solutionfollowed by infection with Ad-LacZ;

[0045]FIGS. 16B and 16C are photographs showing the cross section of themurine bladder of FIG. 16A wherein FIG. 16B was taken at 40× and FIG.16C was taken at 100× magnification;

[0046]FIG. 17A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.4% oxychlorosene solutionfollowed by infection with Ad-LacZ;

[0047]FIGS. 17B and 17C are photographs showing the cross section of themurine bladder of FIG. 17A wherein FIG. 17B was taken at 40× and FIG.17C was taken at 100× magnification;

[0048]FIG. 18A is a photograph showing the luminal surface of a secondmurine bladder after pretreatment with a 0.4% oxychlorosene solutionfollowed by infection with Ad-LacZ;

[0049]FIGS. 18B and 18C are photographs showing the cross section of themurine bladder of FIG. 18A wherein FIG. 18B was taken at 40× and FIG.18C was taken at 100× magnification;

[0050]FIG. 19A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.02% polidocanol solutionfollowed by infection with Ad-LacZ;

[0051]FIGS. 19B and 19C are photographs showing the cross section of themurine bladder of FIG. 19A wherein FIG. 19B was taken at 40× and FIG.19C was taken at 100× magnification;

[0052]FIG. 20A is a photograph showing the luminal surface of a secondmurine bladder after pretreatment with a 0.02% polidocanol solutionfollowed by infection with Ad-LacZ;

[0053]FIGS. 20B and 20C are photographs showing the cross section of themurine bladder of FIG. 20A wherein FIG. 20B was taken at 40× and FIG.20C was taken at 100× magnification;

[0054]FIGS. 21A and 21B are photographs showing the outside and luminalsurfaces, respectively, of a first murine bladder after pretreatmentwith a 0.05% polidocanol solution followed by infection with Ad-LacZ;

[0055]FIGS. 21C and 21D are photographs showing the cross section of themurine bladder of FIG. 21A wherein FIG. 21B was taken at 40× and FIG.21C was taken at 100× magnification;

[0056]FIGS. 22A and 22B are photographs showing the outside and luminalsurfaces, respectively, of a second murine bladder after pretreatmentwith a 0.05% polidocanol solution followed by infection with Ad-LacZ;

[0057]FIGS. 22C and 22D are photographs showing the cross section of themurine bladder of FIG. 22A wherein FIG. 22B was taken at 40× and FIG.22C was taken at 100× magnification;

[0058]FIG. 23A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.2% polidocanol solutionfollowed by infection with Ad-LacZ;

[0059]FIGS. 23B and 23C are photographs showing the cross section of themurine bladder of FIG. 23A wherein FIG. 23B was taken at 40× and FIG.23C was taken at 100× magnification;

[0060]FIG. 24A is a photograph showing the luminal surface of a secondmurine bladder after pretreatment with a 0.2% polidocanol solutionfollowed by infection with Ad-LacZ;

[0061]FIGS. 24B and 24C are photographs showing the cross section of themurine bladder of FIG. 24A wherein FIG. 24B was taken at 40× and FIG.24C was taken at 100× magnification;

[0062]FIG. 25A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.02% n-dodecyl β-D-maltosidesolution followed by infection with Ad-LacZ;

[0063]FIGS. 25B and 25C are photographs showing the cross section of themurine bladder of FIG. 25A wherein FIG. 25B was taken at 40× and FIG.25C was taken at 100× magnification;

[0064]FIG. 26A is a photograph showing the luminal surface of a secondmurine bladder after pretreatment with a 0.02% n-dodecyl β-D-maltosidesolution followed by infection with Ad-LacZ;

[0065]FIGS. 26B and 26C are photographs showing the cross section of themurine bladder of FIG. 26A wherein FIG. 26B was taken at 40× and FIG.26C was taken at 100× magnification;

[0066]FIG. 27A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.05% n-dodecyl β-D-maltosidesolution followed by infection with Ad-LacZ;

[0067]FIGS. 27B and 27C are photographs showing the cross section of themurine bladder of FIG. 27A wherein FIG. 27B was taken at 40× and FIG.27C was taken at 100× magnification;

[0068]FIG. 28A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.05% n-dodecyl β-D-maltosidesolution followed by infection with Ad-LacZ;

[0069]FIGS. 28B and 28C are photographs showing the cross section of themurine bladder of FIG. 28A wherein FIG. 28B was taken at 40× and FIG.28C was taken at 100× magnification;

[0070]FIG. 29A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.2% n-dodecyl β-D-maltosidesolution followed by infection with Ad-LacZ;

[0071]FIGS. 29B and 29C are photographs showing the cross section of themurine bladder of FIG. 29A wherein FIG. 29B was taken at 40× and FIG.29C was taken at 100× magnification;

[0072]FIG. 30A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.2% sodium salt of dedecylbenzenesulfonic acid solution followed by infection with Ad-LacZ; and

[0073]FIGS. 30B and 30C are photographs showing the cross section of themurine bladder of FIG. 30A wherein FIG. 30B was taken at 40× and FIG.30C was taken at 100× magnification.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0074] The present invention is directed to the use of transductionenhancing agents to render the bladder umbrella cell layer moresusceptible to infection with a viral gene delivery vehicle than itwould be without treatment. Exemplary transduction enhancing agentsaccording to the invention include: dodecyl surfactants;dodecylmaltosides; dodecyl alcohol polyoxyethylene ethers (i.e.,polidocanol); and sodium dodecylbenzenesulphonic acid/hypochlorous acidcomplex (i.e., oxychlorosene).

[0075] According to the invention, the luminal surface of the bladdercan be treated with a composition comprising a transduction enhancingagent prior to infection with a viral gene delivery vehicle. The viralgene delivery vehicle can be an oncolytic virus used to treat bladdercancer. Oncolytic viruses for use in practicing the invention include,but are not limited to, adenovirus, herpes simplex virus (HSV),reovirus, vesicular stomatitis virus (VSV), newcastle disease virus,vacinia virus, influenza virus, West Nile virus, coxsackie virus,poliovirus and measles virus. Of particular interest in practicing theinvention are oncolytic viruses that exhibit preferential expression inparticular tissue types (i.e., in the bladder urothelium). An oncolyticadenovirus of this type is disclosed, for example, in Zhang, et al.,“Identification of Human Uroplakin II Promoter and Its Use in theConstruction of CG8840, a Urothelium-specific Adenovirus Variant thatEliminates Established Bladder Tumors in Combination with Docetaxel”,Cancer Research, 62, 3743-3750 (2002) and in co-owned U.S. patentapplication Ser. No. 09/814,292, which is expressly incorporated byreference herein. Chemotherapeutic agents for use in combination therapywith oncolytic viruses are described, for example, in co-owned U.S.patent application Ser. No. 09/814,357, which is expressly incorporatedby reference herein.

[0076] Alternatively, the viral gene delivery vehicle can be any genetherapy delivery vehicle known in the art for use in gene therapy,including, but not limited to, an adenovirus, an adeno-associated virus(AAV), a lentivirus, a retrovirus, a herpes virus, etc. Exemplary genetherapy adenoviral agents are disclosed in U.S. Pat. No. 6,165,779. Thepresent inventors have found that pre-treating mouse bladders withaqueous solutions of various compounds consistently increasedtransduction to greater than 60% of the bladder surface, versus anuntreated percent transduction of no more than 10%.

[0077] In addition to pre-treatment of the bladder surface with thetransduction enhancing agent, the present invention includesco-administration of the viral gene delivery vehicle and thetransduction enhancing agent to the bladder and to co-formulations ofany one of the transduction enhancing agents with a recombinant viralgene delivery vehicle.

[0078] Composition and Chemistry of Reagents Used to Enhance AdenovirusTransduction in the Bladder Epithelium

[0079] Several classes of compounds, surfactants, and pre-made reagentswere tested in order to find those which increased gene transfer ortransduction by a viral gene delivery vehicle in the bladder. Anoncolytic adenovirus, CG884, was used as an exemplary viral gene therapyvehicle. The reagents evaluated can be classified by their physical orchemical properties and structure.

[0080] First, the reagents can be grouped as a single compound or as amixed reagent (i.e., a mixture of compounds). Single compounds evaluatedinclude non-ionic surfactants, alcohols, polymers and ionic surfactants.The ionic surfactants evaluated included: 4% Poloxamer 407 (Pluronic®127); 4% poloxamer 188 (Pluronic® F68); 0.02%-0.5% Polidocanol; 0.1%n-dodecyl-b-D-glucopyranoside (which can also be classified as asugar-based surfactant); 0.02-0.5% n-dodecyl-b-D-maltoside (which canalso be classified as a sugar-based surfactant); 0.1% Tween® 20; 0.1%Triton® X-100; 0.1% Forlan® C-24 (PEG Cholesterol); 0.1%decyl-b-D-maltoside (which can also be classified as a sugar-basedsurfactant); 0.1% 6-cyclohexylhexyl-β-D-maltoside (which can also beclassified as a sugar-based surfactant); and 0.1% Tromboject® (sodiumtetradecyl sulfate).

[0081] Alcohols evaluated include 0.1%-3% benzyl alcohol and 10%-30%ethanol. Polymers evaluated include 0.4% HPMC 2910; 0.4% PVA; 0.4% PVP;and 100 mg/ml Poly-Lysine. Ionic surfactants evaluated include: 0.1%DC-Chol [Cholesteryl 3b-N-(dimethylaminoethyl)carbamate]; 0.2% sodiumsalt of Dodecyl benzenesulfonic acid; and 0.1% sodium dodecyl sulfate.Mixed reagents evaluated include: In vivo GeneSHUTTLE™ (a reagentcomprising DOTAP+Cholesterol available from Qbiogene of Carlsbad,Calif.) and 0.1%-0.4% Oxychlorosene (sodium dodecylbenzenesulphonicacid/hypochlorous acid complex).

[0082] Effect of Ethanol Pretreatment on Adenovirus-Mediated GeneTransfer and Expression in The Bladder Epithelium of Rodents

[0083] A study was conducted to evaluate the effect of ethanolpretreatment on adenovirus-mediated gene transfer and expression in thebladder epithelium of rodents.

[0084] Test Materials

[0085] Ad-βgal virus was made as a frozen formulation using standardconditions known in the art for freezing and formulation of adenovirus.The vehicle for the virus arm was PBS plus 10% glycerol. Pretreatmentagents were 5%, 10%, 15%, 20% and 30% GLP grade ethanol, respectively,in PBS-10% glycerol solution.

[0086] Animals

[0087] 80 female BALB/c mice were used for this study. Female animalsare chosen because of the ease of urethral cannulation and vesicleinstillation. The mice were approximately 10 to 12 weeks on the day ofthe start of the experiment.

[0088] Treatment Regimen

[0089] Animals were assigned to each group as shown in the followingtable. TABLE 1 Effect of Ethanol pretreatment Animals Virus dose Groupper Test Ethanol (particles/ No. group Article Route pretreatmentanimal) Dose Regimen 1 8-10 Vehicle Intravesical — — 100 ml PBS- 10%glycerol on Day 1 2 8-10 Ad-βgal Intravesical — 1.3 × 10¹¹ Ad-βgal onDay 1 3 8-10 Ad-βgal Intravesical  5% ethanol 1.3 × 10¹¹  5% ethanolpretreatment followed by virus administration on Day 1 4 8-10 Ad-βgalIntravesical 10% ethanol 1.3 × 10¹¹ 10% ethanol pretreatment followed byvirus administration on Day 1 5 8-10 Ad-βgal Intravesical 15% ethanol1.3 × 10¹¹ 15% ethanol pretreatment followed by virus administration onDay 1 6 8-10 Ad-βgal Intravesical 20% ethanol 1.3 × 10¹¹ 20% ethanolpretreatment followed by virus administration on Day 1 7 8-10 Ad-βgalIntravesical 25% ethanol 1.3 × 10¹¹ 25% ethanol pretreatment followed byvirus administration on Day 1 8 8-10 Ad-βgal Intravesical 30% ethanol1.3 × 10¹¹ 30% ethanol pretreatment followed by virus administration onDay 1

[0090] For the data in Table 1, the concentration of Ad-βgal virus was1.3×1012 vp/ml as determined by optical density measurements.

[0091] Treatment Procedure

[0092] 1. Animals were anesthetized with isoflurane and a 24 g catheterintroduced through the urethra into the bladder.

[0093] 2. Residual urine was emptied and the bladder was flushed 3 timeswith 100-150 μl each of PBS.

[0094] 3. In test animals, bladders were pretreated for 20 minutes with0.1 ml of 5, 10, 15, 20, 25 or 30% ethanol solution, respectively, andthen rinsed 3 times with 100-150 μl of PBS.

[0095] 4. Ad-βgal viruses diluted in 0.1 ml of PBS-10% glycerol wereadministered intravesically into the bladder and retained in the bladderfor 45 minutes. A knot was placed around the urethral orifice to preventleakage of the virus and to prevent the catheter from dislodging.

[0096] 5. Treatment was stopped by withdrawing the virus and flushingthe bladders 3 times with 100-150 μl of PBS. If the catheter becameclogged, the washing step was avoided so that the virus was flushed outin the urine. However, the use of this procedure may preventdetermination of the viral resident time in the bladder.

[0097] Measurement/Determinations

[0098] The clinical condition of the animals was observed before dosingon the day of treatment and the animals were observed daily during theexperimental period.

[0099] Assessment of β-Galactosidase Activity

[0100] Animals were killed 48 hours after treatment. Bladders werefilled with 0.1 ml whole organ fixative: 2% Neutral buffered formalin,2% glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4. Bladders were thenremoved and immersed in whole organ fixative for 1 hr. Thereafter, thebladders were cut open longitudinally, rinsed (2 mM MgCl₂, 0.1%deoxycholate, 0.2% Triton) for 24 hours at 4° C., and submerged intoX-gal staining solution. Transgene expression in the luminal epitheliumof the longitudinally opened bladders was empirically determined.

[0101] Histopathology

[0102] Bladders fixed in whole organ fixative were sectioned and stainedwith hematoxylin-eosin for histologic examination.

[0103] Results

[0104] Pretreatment of the luminal bladder surface with variousconcentrations of ethanol (i.e., 15%, 20%, 25%, and 30 wt. %) for 20minutes resulted in 10-20% transduction. FIGS. 1-4 show transduction ofmurine bladders after pretreatment with ethanol. FIGS. 1A and 1B arephotographs showing a murine bladder after pretreatment with a 15%ethanol solution followed by infection with Ad-LacZ. FIG. 1A shows theoutside surface of the bladder and FIG. 1B shows the luminal bladdersurface. FIGS. 1C and 1D are photographs showing a murine bladder afterpretreatment with a 20% ethanol solution followed by infection withAd-LacZ. FIG. 1C shows the outside surface of the bladder and FIG. 1Dshows the luminal bladder surface. FIGS. 1E and 1F are photographsshowing a murine bladder after pretreatment with a 25% ethanol solutionfollowed by infection with Ad-LacZ. FIG. 1E shows the outside surface ofthe bladder and FIG. 1F shows the luminal bladder surface. FIGS. 1G and1H are photographs showing a murine bladder after pretreatment with a30% ethanol solution followed by infection with Ad-LacZ. FIG. 1G showsthe outside surface of the bladder and FIG. 1H shows the luminal bladdersurface. As can be seen from FIG. 1, higher concentrations of ethanolresulted in greater levels of transduction as measured by staining.

[0105]FIG. 2A is a photograph showing a cross section of a murinebladder control (i.e., no pretreatment). FIGS. 2B and 2C are photographsshowing the cross section of a murine bladder after pretreatment with a30% ethanol solution followed by infection with Ad-LacZ.

[0106] FIGS. 3A-3F are photographs showing the cross section of threemurine bladders after pretreatment with a 25% ethanol solution followedby infection with Ad-LacZ. FIGS. 3A and 3B are photographs showing thecross-section of the first murine bladder, FIGS. 3C and 3D arephotographs showing the cross-section of the second murine bladder, andFIGS. 3E and 3F are photographs showing the cross-section of the thirdmurine bladder. FIGS. 3A, 3C and 3E were taken at 40× and FIGS. 3B, 3Dand 3F were taken at 100× magnification.

[0107] FIGS. 4A-4F are photographs showing the cross section of threemurine bladders after pretreatment with a 30% ethanol solution followedby infection with Ad-LacZ. FIGS. 4A and 4B are photographs showing thecross-section of the first murine bladder, FIGS. 4C and 4D arephotographs showing the cross-section of the second murine bladder, andFIGS. 4E and 4F are photographs showing the cross-section of the thirdmurine bladder. FIGS. 4A, 4C and 4E were taken at 40× and FIGS. 4B, 4Dand 4F were taken at 100× magnification.

[0108] Effect of Chemical Agent Pretreatment on Adenovirus-Mediated GeneTransfer and Expression in The Bladder Epithelium of Rodent

[0109] A study was conducted to evaluate the effect of chemical agentpretreatment on adenovirus-mediated gene transfer and expression in thebladder epithelium of rodents.

[0110] Test Materials

[0111] Ad-βgal virus was made at CGI, as a frozen formulation usingstandard conditions known in the art for freezing and formulation ofadenovirus. The vehicle for the virus arm was PBS plus 10% glycerol.

[0112] Animals

[0113] 152 female BALB/c mice were used this study. Female animals werechosen because of the ease of urethral cannulation and vesicleinstillation. The mice were approximately 10 to 12 weeks on the day ofthe start of the experiment.

[0114] Treatment Regimen

[0115] Animals were assigned to each group shown in the following table.The route of administration of the chemical agent and virus wasintravesical. TABLE 2 Effect of Chemical Agent Pretreatment AnimalsVirus dose Group per Test (particles/ No. group Article Chemical agentanimal) Dose Regimen 1 2 Vehicle 4% Poloxamer 407 — 100 ml of 4%(Pluronic 127) Poloxamer 407 (Pluronic 127) in PBS-10% glycerol on Day 12 6-8 Ad-βgal 4% Poloxamer 407 1.3 × 10¹¹ 4% Poloxomer 407 (Pluronic127) (Pluronic 127) pretreatment followed by virus administration on Day1 3 2 Vehicle 4% Poloxamer 188 100 ml of 4% (Pluronic F68) Poloxamer 188(Plluronic F68) in PBS-10% glycerol on Day 1 4 6-8 Ad-βgal 4% Poloxamer188 1.3 × 10¹¹ 4% Poloxomer 188 (Pluronic F68) (Pluronic F68)pretreatment following by virus administration on Day 1 5 2 VehicleLipofectamine 100 ml of 2000 Lipofectamine 2000 (20 mg/ml) in PBS 10%glycerol. 6 6-8 Ad-βgal Lipofectamine 0.65 × 10¹¹  1.25 mg of 2000Lipofectamine 2000 mixed with virus administration on Day 1 7 2 Vehicle3% Benzyl 100 μl of 3% Benzyl Alcohol Alcohol in PBS-10% glycerol on Day1 8 6-8 Ad-βgal 3% Benzyl   1 × 10¹¹ 3% Benzyl Alcohol Alcoholpretreatment followed by virus administration on Day 1 9 2 Vehicle 0.2%0.2% Oxychlorosene Oxychlorosene in PBS-10% glycerol on Day 1 10 6-8Ad-βgal 0.2% 1.3 × 10¹¹ 0.2% Oxychlorosene Oxychlorosene pretreatment(only wash) followed by virus administration on Day 1 11 6-8 Ad-βgal0.2% 1.3 × 10¹¹ 0.2% Oxychlorosene Oxychlorosene pretreatment (5 min)followed by virus administration on Day 1 12 6-8 Ad-βgal 0.2% 1.3 × 10¹¹0.2% Oxychlorosene Oxychlorosene pretreatment (15 min) followed by virusadministration on Day 1 13 2 Vehicle 0.05% Polidocanol 0.05% Polidocanolin PBS-10% glycerol on Day 1 14 6-8 Ad-βgal 0.05% Polidocanol 1.3 × 10¹¹0.05% Polidocanol pretreatment followed by virus administration on Day 115 2 Vehicle 0.1% DC-Chol 0.1% DC-Chol in PBS-10% glycerol on Day 1 166-8 Ad-βgal 0.1% DC-Chol 1.3 × 10¹¹ 0.1% DC-Chol pretreatment followedby virus administration on Day 1 17 2 Vehicle In vivo Gene 4 mM solutionin PBS Shuttle (DOTAP + Cholesterol) 18 6-8 Ad-βgal In vivo Gene 0.65 ×10¹¹  4 mM of In vivo Gene Shuttle (DOTAP + Shuttle mixed withCholesterol) virus. Administration on Day 1. (Dilute 60 ml of Lipid with90 ml of water. Then add 150 ul of Ad-bgal) 19 2 Vehicle 0.5%Polidocanol 0.5% Polidocanol in PBS-10% glycerol on Day 1 20 6-8 Ad-βgal0.5% Polidocanol 1.3 × 10¹¹ 0.5% Polidocanol pretreatment followed byvirus administration on Day 1 21 2 Vehicle 0.4% HPMC 2910 0.4% HPMC 2910in PBS-10% glycerol on Day 1 22 6-8 Ad-βgal 0.4% HPMC 2910 0.5 × 10¹¹0.8% HPMC 2910 mixed with an equal volume of the virus and thenadministered on Day 1 23 2 Vehicle 100 mg/ml Poly- 100 ug/ml Poly-LysineLysine in PBS-10% glycerol on Day 1 24 6-8 Ad-βgal 100 mg/ml Poly- 0.5 ×10¹¹ 200 ug/ml Poly-Lysine Lysine mixed with an equal volume of thevirus and then administered on Day 1 25 2 Vehicle 0.1% n-dodecyl-b- 0.1%n-dodecyl-b-D D glucopyranoside glucopyranoside in PBS-10% glycerol onDay 1 26 6-8 Ad-βgal 0.1% n-dodecyl-b-   1 × 10¹¹ 0.1% n-dodecyl-b-D Dglucopyranoside glucopyranoside pretreatment followed by virusadministration on Day 1 27 2 Vehicle 0.4% PVA 0.4% PVA in PBS- 10%glycerol on Day 1 28 6-8 Ad-βgal 0.4% PVA 0.5 × 10¹¹ 0.8% PVA mixed withan equal volume of the virus and then administered on Day 1 29 2 Vehicle0.4% PVP 0.4% PVP in PBS- 10% glycerol on Day 1 30 6-8 Ad-βgal 0.4% PVP0.5 × 10¹¹ 0.8% PVP mixed with an equal volume of the virus and thenadministered on Day 1 31 2 Vehicle 0.1% Cholesterol- 0.1% Cholesterol-Cyclodextrin Cyclodextrin reagent reagent in PBS-10% glycerol on Day 132 6-8 Ad-βgal 0.1% Cholesterol- 0.5 × 10¹¹ 0.2% Cholesterol-Cyclodextrin Cyclodextrin reagent reagent mixed with an equal volume ofthe virus and then administered on Day 1 33 2 Vehicle 0.05% n-Dodecyl0.05% n-Dodecyl b-D- b-D-Maltoside Maltoside in PBS- 10% glycerol on Day1 34 6-8 Ad-βgal 0.05% n-Dodecyl   1 × 10¹¹ 0.05% n-Dodecyl b-D-b-D-Maltoside Maltoside pretreatment followed by virus administration onDay 1 35 2 Vehicle 0.3% Benzyl 100 μl of 0.3% Benzyl Alcohol Alcohol inPBS-10% glycerol on Day 1 36 6-8 Ad-βgal 0.3% Benzyl   1 × 10¹¹ 0.3%Benzyl Alcohol Alcohol pretreatment followed by virus administration onDay 1 37 2 Vehicle 0.1% Benzyl 100 μl of 0.1% Benzyl Alcohol Alcohol inPBS-10% glycerol on Day 1 38 6-8 Ad-βgal 0.1% Benzyl   1 × 10¹¹ 0.1%Benzyl Alcohol Alcohol pretreatment followed by virus administration onDay 1 39 2 Vehicle 0.1% 0.1% Oxychlorosene Oxychlorosene in PBS-10%glycerol on Day 1 40 6-8 Ad-βgal 0.1%   1 × 10¹¹ 0.1% OxychloroseneOxychlorosene pretreatment (5 min) followed by virus administration onDay 1 41 2 Vehicle 0.4% 0.4% Oxychlorosene Oxychlorosene in PBS-10%glycerol on Day 1 42 6-8 Ad-βgal 0.4%   1 × 10¹¹ 0.4% OxychloroseneOxychlorosene pretreatment (5 min) followed by virus administration onDay 1 43 2 Vehicle 0.02% Polidocanol 0.02% Polidocanol in PBS-10%glycerol on Day 1 44 6-8 Ad-βgal 0.02% Polidocanol   1 × 10¹¹ 0.02%Polidocanol pretreatment followed by virus administration on Day 1 45 2Vehicle 0.2% Polidocanol 0.2% Polidocanol in PBS-10% glycerol on Day 146 6-8 Ad-βgal 0.2% Polidocanol   1 × 10¹¹ 0.2% Polidocanol pretreatmentfollowed by virus administration on Day 1 47 2 Vehicle 0.02% n-Dodecyl0.02% n-Dodecyl b-D- b-D-Maltoside Maltoside in PBS- 10% glycerol on Day1 48 6-8 Ad-βgal 0.02% n-Dodecyl   1 × 10¹¹ 0.02% n-Dodecyl b-D-b-D-Maltoside Maltoside pretreatment followed by virus administration onDay 1 49 2 Vehicle 0.2% n-Dodecyl b- 0.2% n-Dodecyl b-D- D-MaltosideMaltoside in PBS- 10% glycerol on Day 1 50 6-8 Ad-βgal 0.2% n-Dodecyl b-  1 × 10¹¹ 0.2% n-Dodecyl b-D D-Maltoside Maltoside pretreatmentfollowed by virus administration on Day 1 51 2 Vehicle 0.2% sodium salt0.2% sodium salt of of Dodecyl Dodecyl benzenesulfonic benzenesulfonicacid acid in PBS-10% glycerol on Day 1 52 6-8 Ad-βgal 0.2% sodium salt  1 × 10¹¹ 0.2% sodium salt of of Dodecyl Dodecyl benzenesulfonicbenzenesulfonic acid acid pretreatment followed by virus administrationon Day 1 53 2 Vehicle 0.1% sodium 0.1% sodium dodecyl dodecyl sulphatesulphate in PBS-10% glycerol on Day 1 54 6-8 Ad-βgal 0.1% sodium   1 ×10¹¹ 0.1% sodium dodecyl dodecyl sulphate sulphate pretreatment followedby virus administration on Day 1 55 2 Vehicle 0.1% Tween 20 0.1% Tween20 in PBS-10% glycerol on Day 1 56 6-8 Ad-βgal 0.1% Tween 20   1 × 10¹¹0.1% Tween 20 pretreatment followed by virus administration on Day 1 572 Vehicle 0.1% Triton X-100 0.1% Triton X-100 in PBS-10% glycerol on Day1 58 6-8 Ad-βgal 0.1% Triton X-100   1 × 10¹¹ 0.1% Triton X-100pretreatment followed by virus administration on Day 1 59 2 Vehicle 0.1%Forlan C-24 0.1% Forlan C-24 in (PEG Cholesterol) PBS-10% glycerol onDay 1 60 6-8 Ad-βgal 0.1% Forlan C-24   1 × 10¹¹ 0.1% Forlan C-24 (PEGCholesterol) pretreatment followed by virus administration on Day 1 61 2Vehicle 0.1% Decyl-b-D- 0.1% Decyl-b-D- Maltoside Maltoside in PBS- 10%glycerol on Day 1 62 6-8 Ad-βgal 0.1% Decyl-b-D-   1 × 10¹¹ 0.1%Decyl-b-D- Maltoside Maltoside pretreatment followed by virusadministration on Day 1 63 2 Vehicle 0.1% 6- 0.1% 6- Cyclohexylhexyl-Cyclohexylhexyl-b-D- b-D-Maltoside Maltoside in PBS- 10% glycerol on Day1 64 6-8 Ad-βgal 01% 6-   1 × 10¹¹ 0.1% 6- Cyclohexylhexyl-Cyclohexylhexyl-b-D- b-D-Maltoside Maltoside pretreatment followed byvirus administration on Day 1 65 2 Vehicle 0.1% Tromboject 0.1%Tromboject in (Sodium PBS-10% glycerol on Tetradecyl Sulfate) Day 1 666-8 Ad-βgal 0.1% Tromboject   1 × 10¹¹ 0.1% Tromboject (Sodiumpretreatment followed Tetradecyl Sulfate) by virus administration on Day1 67 2 Vehicle 0.1% Phenyl B-D- 0.1% Phenyl B-D- GlucopyranosideGlucopyranoside in PBS-10% glycerol on Day 1 68 6-8 Ad-βgal 0.1% PhenylB-D-   1 × 10¹¹ 0.1% Phenyl B-D- Glucopyranoside Glucopyranosidepretreatment followed by virus administration on Day 1 69 2 Vehicle 0.1%Sucrose 0.1% Sucrose Monolaurate Monolaurate in PBS- 10% glycerol on Day1 70 6-8 Adβgal 0.1% Sucrose   1 × 10¹¹ 0.1% Sucrose MonolaurateMonolaurate pretreatment followed by virus administration on Day 1 71 2Vehicle 0.1% 1-O-dodecyl- 0.1% 1-O-dodecyl- rac-glycerol rac-glycerol inPBS- 10% glycerol on Day 1 72 6-8 Ad-βgal 0.1% 1-O-dodecyl-   1 × 10¹¹0.1% 1-O-dodecyl- rac-glycerol rac-glycerol pretreatment followed byvirus administration on Day 1

[0116] The concentration of Ad-βgal virus for the data in Table 2 was1.3×1012 vp/ml (1st preparation, particle: PFU: 30) and 1×10¹² vp/ml(2^(nd) preparation, particle: PFU: 30) as determined by optical densitymeasurements.

[0117] Treatment Procedure

[0118] 1. The animals were anesthetized with isoflurane and a 24 gcatheter was introduced through the urethra into the bladder.

[0119] 2. Residual urine was emptied and the bladder was flushed 3 timeswith 100 μl each of PBS.

[0120] 3. Based on the reagent being tested, bladder pretreatment wasperformed as follows:

[0121] Poloxomer 407 procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and gave one additional wash.Performed 3 times PBS wash prior to virus instillation.

[0122] Poloxomer 188 procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 minutes and gave one additional wash.Performed 3 times PBS wash prior to virus instillation.

[0123] Lipofectamine 2000 procedure: Added 5 μl of stock Lipofectamine(1 mg/ml) to 195 μl of PBS-10% glycerol. Mixed with an equal volume ofAd-βgal virus and incubated for 15 minutes. Administered 100 μl of themixture intravesically and retained in the bladder for 30 minutes.

[0124] Benzyl Alcohol procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 15 minutes and then gave one additionalwash. Performed 3 times PBS wash prior to virus instillation.

[0125] Oxychlorosene procedure: Washing performed as mentioned in thedose regimen (i.e., 3 washes of 100 μl each, one wash but retained for 5min., one wash but retained for 15 min). Performed 3 times PBS washprior to virus instillation.

[0126] Polidocanol procedure: Washed 2 times with 100 μl each. Retainedthe 3^(rd) wash for 5 min and then gave one additional wash. Performed 3times PBS wash prior to virus instillation.

[0127] DC-Cho procedure: Washed 2 times with 100 μl each. Retained the3^(rd) wash for 5 min and then gave one additional wash. Performed 3times PBS wash prior to virus instillation.

[0128] 0.4% HPMC 2910 procedure: No pretreatment. An equal volume of thevirus was mixed with 0.8% solution of HPMC2910 and the mixture wasinstilled into the bladder for 30 minutes.

[0129] 100 mg/ml Poly-Lysine procedure: No pretreatment. An equal volumeof the virus was mixed with 100 mg/ml solution of Poly-Lysine and themixture was instilled into the bladder for 30 minutes.

[0130] 0.4% polyvinyl alcohol (PVA) procedure: No pretreatment. An equalvolume of the virus was mixed with 0.8% solution of PVA and the mixturewas instilled into the bladder for 30 minutes.

[0131] n-dodecyl-p-D glucopyranoside procedure: Washed 2 times with 100μl each. Retained the 3^(rd) wash for 5 min and then gave one additionalwash. Performed 3 times PBS wash prior to virus instillation.

[0132] 0.4% PVP procedure: No pretreatment. An equal volume of the viruswas mixed with 0.8% solution of PVP and the mixture was instilled intothe bladder for 30 min.

[0133] 0.1% cholesterol-cyclodextrin reagent procedure: No pretreatment.An equal volume of the virus was mixed with 0.2% solution ofCholesterol-Cyclodextrin and the mixture was instilled into the bladderfor 30 minutes.

[0134] n-dodecyl-β-D-maltoside procedure: Washed 2 times with 100 μleach. Retained the 3^(rd) wash for 5 min and then gave one additionalwash. Performed 3 times PBS wash prior to virus instillation.

[0135] Sodium salt of dodecyl benzenesulfonic acid procedure: Washed 2times with 100 μl each. Retained the 3^(rd) wash for 5 min and then gaveone additional wash. Performed 3 times PBS wash prior to virusinstillation.

[0136] 0.1% sodium dodecyl sulphate procedure: Wash 2 times with 100 μleach. Retained the 3^(rd) wash for 5 min and then gave one additionalwash. Performed 3 times PBS wash prior to virus instillation.

[0137] 0.1% Tween 20 procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and then gave one additional wash.Performed 3 times PBS wash prior to virus instillation.

[0138] 0.1% Triton® X-100 procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and then gave one additional wash.Perform 3 times PBS wash prior to virus instillation.

[0139] 0.1% Forlan C-24 procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and then gave one additional wash.Performed 3 times PBS wash prior to virus instillation.

[0140] 0.1% decyl-b-D-maltoside procedure: Washed 2 times with 100 μleach. Retained the 3^(rd) wash for 5 min and then gave one additionalwash. Performed 3 times PBS wash prior to virus instillation.

[0141] 0.1% 6-cyclohexylhexyl-b-D-maltoside procedure: Washed 2 timeswith 100 μl each. Retained the 3^(rd) wash for 5 min and then gave oneadditional wash. Performed 3 times PBS wash prior to virus instillation.

[0142] 0.1% sodium tetradecyl sulfate (Tromboject®, Omega LaboratoriesLtd.) procedure: Washed 2 times with 100 μl each. Retained the 3^(rd)wash for 5 min and then gave one additional wash. Performed 3 times PBSwash prior to virus instillation.

[0143] 0.1% phenyl-β-D-glucopyranoside procedure: Washed 2 times with100 μl each. Retained the 3^(rd) wash for 5 min and then gave oneadditional wash. Performed 3 times PBS wash prior to virus instillation.0.1% sucrose monolaurate procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and then gave one additional wash.Performed 3 times PBS wash prior to virus instillation. 0.1%1-O-dodecyl-rac-glycerol procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and then gave one additional wash.Performed 3 times PBS wash prior to virus instillation.

[0144] In vivo geneSHUTTLE™ procedure. Mixed 4 mM of In vivogeneSHUTTLE™ with virus. Administration on Day 1. Diluted 60 ml of Lipidwith 90 ml of water. Then added 150 μl of Ad-βgal. 4. Virus treatment(45 min) stopped by withdrawing the virus and flushing the bladders 3times with 100 μl of PBS.

[0145] Measurement/Determinations

[0146] The clinical condition of the animals were observed before dosingon the day of treatment, and animals were observed daily during theexperimental period.

[0147] Assessment of β-galactosidase Activity

[0148] Animals were killed 48 hours after treatment. The bladders werefilled with 0.1-ml whole organ fixative: 2% Neutral buffered formalin,2% glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4. The bladders were thenremoved and immersed in whole organ fixative for 1 hour. Thereafter,each bladder was cut open longitudinally, rinsed (in 2 mM MgCl₂, 0.1%deoxycholate, 0.2% Triton) for 24 hours at 4° C., and submerged intoX-gal staining solution. Transgene expression in the luminal epitheliumof the longitudinally opened bladders was empirically determined.

[0149] Histopathology

[0150] Bladders fixed in whole organ fixative were sectioned and stainedwith hematoxylin-eosin for histologic examination.

[0151] Results

[0152] The results of the above experiments can be summarized asfollows:

[0153] Pre-treatment of the bladder with 4% Poloxamer 407 (Pluronic 127)for 5 minutes resulted in <5% transduction.

[0154] Treatment of the bladder with a lipofectamine and virus mixture(no pretreatment) resulted in <5% transduction.

[0155] Treatment of the bladder with an In vivo geneSHUTTLE™ and virusmix (no bladder pretreatment) resulted in <5% transduction.

[0156] A pre-treatment of the bladder with 0.1% oxychlorosene for 5minutes resulted in >90% transduction of the urothelium. Thepathologists report indicated mild submucosal edema with intactepithelial layer.

[0157] A pre-treatment of the bladder with 0.2% oxychlorosene for 5minutes resulted in >90% transduction of the urothelium. Thepathologists report indicated minimal submucosal edema and perivascularlymphocytes.

[0158] A pre-treatment of the bladder with 0.2% oxychlorosene for 15minutes resulted in >90% transduction of Urothelium. The pathologistsreport indicated focal severe ulceration with suppurtative exudate,hemorrhage and edema in the submucosa.

[0159] A pre-treatment of the bladder with 0.4% oxychlorosene for 5minutes resulted in >90% transduction of Urothelium. The pathologistsreport indicated moderate submucosal edema with focal large ulcer.

[0160] A pre-treatment of the bladder with 0.02% polidocanol for 5minutes resulted in 10-20% transduction of the urothelium. Thepathologists report indicated an intact mucosa.

[0161] A pre-treatment of the bladder with 0.05% polidocanol for 5minutes resulted in 30-40% transduction of the urothelium. Thepathologists report indicated minimal submucosal edema.

[0162] A pre-treatment of the bladder with 0.2% polidocanol for 5minutes resulted in 50-80% transduction of Urothelium. The pathologistsreport indicated erosions and focal ulcer as well as mucosal compromise.

[0163] A pre-treatment of the bladder with 0.02% n-dodecyl P-D-maltosidefor 5 minutes resulted in 50-80% transduction of the urothelium. Thepathologists report indicated no significant lesions.

[0164] A pre-treatment of the bladder with 0.05% n-dodecyl β-D-maltosidefor 5 minutes resulted in >90% transduction of the urothelium. Thepathologists report indicated no significant lesions.

[0165] A pre-treatment of the bladder with 0.2% n-dodecyl β-D-maltosidefor 5 minutes resulted in >90% transduction of the urothelium. Thepathologists report indicated erosions, focal ulcer, moderate submucosaledema with mucosal compromise.

[0166] A pre-treatment of the bladder with 0.2% dodecyl benzenesulfonicacid for 5 minutes resulted in 20-40% transduction of the urothelium.

[0167] As can be seen from the above results, several single compoundsand one mixed reagent showed significantly increased transduction asmeasured by the levels of final blue stain (LacZ). Several other singlecompounds resulted in enhanced but smaller levels of transduction. Anethanol pre-treatment was used as a reference to validate each chemicaltested. Even with an ethanol percentage as high as 30%, only 10-20%transduction was observed. The “strong responders” were thosetransduction enhancing agents which exhibited significantly better(i.e., 70-90% staining) than the ethanol pre-treatment controls, whichexhibited 10-20% staining. The weak responders had significantly lessstained area compared to the ethanol control group.

[0168] The strongest response (i.e., highest level of transduction) wasobserved following pretreatment of the bladder surface with: 0.02%-0.5%polidocanol; 0.02 -0.5% n-dodecyl-b-D-maltoside; 0.1%6-cyclohexylhexyl-b-D-maltoside; 0. %-0.4% oxychlorosene; 0.2% sodiumsalt of dodecyl benzenesulfonic acid; and 0.1% sodium dodecyl sulphate.

[0169] The “weak responders” included 0.1% decyl-b-D-maltoside and 0.1%Triton® X-100.

[0170] Although not wishing to be bound by theory, the mechanism ofaction can be hypothesized by analyzing the physical and chemicalproperties of successful transduction enhancing reagents. Thetransduction enhancing reagent in general is a surfactant. Thesurfactant can be ionic or non-ionic. The surfactant preferably has bothhydrophilic and lipophilic sections. The hydrophilic portion of themolecule contributes to water solubility while the lipophilic (i.e.,hydrophobic) portion helps molecular interactions with lipids. Thehydrophilic/lipophilic balance or HLB ratio is an indication of therelative size of each part of the molecule.

[0171] Sugar Based Surfactants (Saccharides)

[0172] The transduction enhancing agent according to the invention canbe a sugar (e.g., a mono-, di-, or poly-saccharide) having a lipophilicsubstituent. The transduction enhancing agent can be any mono-, di-, orpoly-saccharide having a lipophilic substituent. According to apreferred embodiment of the invention, the transduction enhancing agentis a di-saccharide having a lipophilic substituent. Exemplarydi-saccharides include maltose or sucrose. Other di-saccharides havinglipophilic substituents, however, can also be used including lactose,isomaltose, trehalose or cellobiose.

[0173] The lipophilic substituent can be linear (e.g., a straight chainn-alkane or alkene) or non-linear (e.g., cyclic or branched chainalkanes or alkenes). The lipophilic substituent can also be an alkanoicacid residue. The length of the lipophilic substituent can be varied toachieve the desired hydrophilic-lipophilic balance. Tests on variousmaltoside substituted compounds indicated that a sufficient lipophiliclength resulted in improved transduction efficacy. For example, bothn-dodecyl-β-D-maltoside and 6-cyclohexylhexyl-β-D-maltoside increasedtransduction significantly. In contrast, n-decyl-β-D-maltoside had onlya slight effect on transduction.

[0174] Results for bladder pretreatment with n-dodecyl-β-D-maltoside areshown in FIGS. 25-29. FIG. 25A is a photograph showing the luminalsurface of a first murine bladder after pretreatment with a 0.02%n-dodecyl β-D-maltoside solution followed by infection with Ad-LacZ.FIGS. 25B and 25C are photographs showing the cross section of themurine bladder of FIG. 25A. FIG. 25B was taken at 40× and FIG. 25C wastaken at 100× magnification. FIG. 26A is a photograph showing theluminal surface of a second murine bladder after pretreatment with a0.02% n-dodecyl β-D-maltoside solution followed by infection withAd-LacZ. FIGS. 26B and 26C are photographs showing the cross section ofthe murine bladder of FIG. 26A. FIG. 26B was taken at 40× and FIG. 26Cwas taken at 100× magnification. FIG. 27A is a photograph showing theluminal surface of a first murine bladder after pretreatment with a0.05% n-dodecyl P-D-maltoside solution followed by infection withAd-LacZ. FIGS. 27B and 27C are photographs showing the cross section ofthe murine bladder of FIG. 27A. FIG. 27B was taken at 40× and FIG. 27Cwas taken at 100× magnification. FIG. 28A is a photograph showing theluminal surface of a first murine bladder after pretreatment with a0.05% n-dodecyl P-D-maltoside solution followed by infection withAd-LacZ. FIGS. 28B and 28C are photographs showing the cross section ofthe murine bladder of FIG. 28A. FIG. 28B was taken at 40× and FIG. 28Cwas taken at 100× magnification. FIG. 29A is a photograph showing theluminal surface of a first murine bladder after pretreatment with a 0.2%n-dodecyl β-D-maltoside solution followed by infection with Ad-LacZ.FIGS. 29B and 29C are photographs showing the cross section of themurine bladder of FIG. 29A. FIG. 29B was taken at 40× and FIG. 29C wastaken at 100× magnification.

[0175] The chemical formula for n-dodecyl-β-D-maltoside andn-decyl-β-D-maltoside is given below:

[0176] where n is 11 and 9, respectively. The chemical formula for6-cyclohexylhexyl-β-D-maltoside is:

[0177] where n is 6.

[0178] The transduction experiments demonstrated that a small reductionin the size of the lipophilic side chain (i.e., CH₂—CH₂) can limit theefficacy of the molecule for transduction enhancement to a great degree.It is important to note that all of the above compounds had goodsolubility in both water and PBS buffer.

[0179] Compounds in this class of surfactants having a shorterhydrophilic moiety were also evaluated. The results forn-dodecyl-β-D-glucopyranoside showed little or no enhancement oftransduction. The chemical formula for n-dodecyl-β-D-glucopyranoside is:

[0180] where n is 11. While not wishing to be bound by theory, therelative sizes of the hydrophilic and lipophilic portions of themolecule appear to influence transduction enhancement. Therefore,shorter chain n-alkyl-β-D-glucopyranosides (e.g.,n-hexyl-β-D-glucopyranoside) may exhibit improved transduction.

[0181] Any mono-, di-, or poly-saccharide having a lipophilicsubstituent can be used as a transduction enhancing agent according tothe invention. Exemplary di-saccharide compounds include sucrose,lactose, maltose, isomaltose, trehalose, and cellobiose. The lipophilicsubstituent preferably comprises an alkyl or alkenyl group. According toa preferred embodiment of the invention, the lipophilic substituent isan alkanoic acid residue.

[0182] Although the β-forms of the mono- and di-saccharides aredescribed above, the α-forms of these and other mono-, di-, orpoly-saccharide compounds can also be used according to the invention.Exemplary α-saccharide transduction enhancing agents according to theinvention include n-dodecyl-α-D-maltoside, n-hexyl-α-D-glucopyranosideand 6-cyclohexylhexyl-a-D-maltoside. Additionally, either the D- orL-forms of the mono-, di-, or poly-saccharides may be used astransduction enhancing agents according to the invention.

[0183] Ionic Alkyl Surfactants

[0184] Ionic alkyl surfactants can also be used as a transductionenhancing compounds according to the invention. Exemplary ionic alkylsurfactants include sodium dodecyl sulfate which has a formularepresented by:

[0185] Another exemplary ionic surfactant is the sodium salt ofdodecyl-benzenesulfonic acid which has a chemical formula representedby:

[0186] Surfactants of the above type were evaluated and were found toexhibit enhanced transduction comparable to the non-ionic reagents setforth above. These results are shown in FIG. 30 for dodecylbenzenesulfonic acid sodium salt. As can be seen by FIGS. 30A-30C,dodecyl benzenesulfonic acid sodium salt, enhanced the transduction ofAd-LacZ in murine bladders. FIG. 30A is a photograph showing the luminalsurface of a first murine bladder after pretreatment with a 0.2% sodiumsalt of dedecyl benzenesulfonic acid solution followed by infection withAd-LacZ. FIGS. 30B and 30C are photographs showing the cross section ofthe murine bladder of FIG. 30A. FIG. 30B was taken at 40× and FIG. 30Cwas taken at 100× magnification.

[0187] The ionic alkyl surfactants consist of two portions, ahydrophilic portion and a lipophilic portion. The arrangement of theseportions of the molecule is similar to the sugar-based enhancing agentsdescribed above. According to the invention, compounds similar to thoseset forth above and having variations in alkyl substitution can also beused.

[0188] Alkyl(ether) Alcohols

[0189] Also according to the invention, an alkyl ether compound can beused as a transduction enhancing compound. Polidocanol, an alkyl etherhaving the following chemical formula:

C₁₂H₂₆—O—(CH₂—CH₂—O)⁻⁹

[0190] and a total formula of ˜C₃₀H₆₂O₁₀, was evaluated. The polidocanolused in the evaluation was sold under the name Thesit®, which is aregistered trademark of Desitin-Werk, Carl Klinke GmbH, Hamburg,Germany). There are several other chemical names for polidocanol such aspolyethyleneglycoldodecyl ether [9002-92-0], lauryl alcohol, andmacrogol lauryl ether.

[0191] Results for pretreatment of the bladder surface with variousconcentrations of polidocanol are shown in FIGS. 19-24. Results forpretreatment of the bladder surface with 0.02% polidocanol are shown inFIGS. 19 and 20. FIG. 19A is a photograph showing the luminal surface ofa first murine bladder after pretreatment with a 0.02% polidocanolsolution followed by infection with Ad-LacZ. FIGS. 19B and 19C arephotographs showing the cross section of the murine bladder of FIG. 19A.FIG. 19B was taken at 40× and FIG. 19C was taken at 100× magnification.FIG. 20A is a photograph showing the luminal surface of a second murinebladder after pretreatment with a 0.02% polidocanol solution followed byinfection with Ad-LacZ. FIGS. 20B and 20C are photographs showing thecross section of the murine bladder of FIG. 20A. FIG. 20B was taken at40× and FIG. 20C was taken at 100× magnification.

[0192] Results for pretreatment of the bladder surface with 0.05%polidocanol are shown in FIGS. 21 and 22. FIGS. 21A and 21B arephotographs showing the outside and luminal surfaces, respectively, of afirst murine bladder after pretreatment with a 0.05% polidocanolsolution followed by infection with Ad-LacZ. FIGS. 21C and 21D arephotographs showing the cross section of the murine bladder of FIG. 21A.FIG. 21B was taken at 40× and FIG. 21C was taken at 100× magnification.FIGS. 22A and 22B are photographs showing the outside and luminalsurfaces, respectively, of a second murine bladder after pretreatmentwith a 0.05% polidocanol solution followed by infection with Ad-LacZ.FIGS. 22C and 22D are photographs showing the cross section of themurine bladder of FIG. 22A. FIG. 22B was taken at 40× and FIG. 22C wastaken at 100× magnification;

[0193] Results for pretreatment of the bladder surface with 0.2%polidocanol are shown in FIGS. 23 and 24. FIG. 23A is a photographshowing the luminal surface of a first murine bladder after pretreatmentwith a 0.2% polidocanol solution followed by infection with Ad-LacZ.FIGS. 23B and 23C are photographs showing the cross section of themurine bladder of FIG. 23A. FIG. 23B was taken at 40× and FIG. 23C wastaken at 100× magnification. FIG. 24A is a photograph showing theluminal surface of a second murine bladder after pretreatment with a0.2% polidocanol solution followed by infection with Ad-LacZ. FIGS. 24Band 24C are photographs showing the cross section of the murine bladderof FIG. 24A. FIG. 24B was taken at 40× and FIG. 24C was taken at 100×magnification.

[0194] Triton® X-100, having a general formula of:

[0195] wherein x=10 was also evaluated and was also found to enhancetransduction. A similar compound having a cyclohexane ring rather than abenzene ring can also be used as a transduction enhancing agentaccording to the invention. This compound has the following chemicalstructure:

[0196] wherein x=10. Compounds of the above type wherein x is anypositive integer can also be used according to the invention.

[0197] Similar alkyl(ether) compounds having the general structure of:

[0198] are also commercially available. The trade name for thesecompounds is “Brij”. The compound shown above is designated “Brij 56”.Brij 56 has the chemical formula C₂₀H₄₂O₅. Another commerciallyavailable compound, “Brij 58”, has the chemical formula C₁₆H₁₁₄O₂₁.

[0199] Any of the above mentioned alkyl(ether) compounds can be used astransduction enhancing agents according to the invention.

[0200] Sodium Oxychlorosene

[0201] A composition comprising a sodium salt of dodecylbenzenesulfonicacid and hypochlorous acid (i.e., sodium oxychlorosene) at a pH of about6.5 to 6.9 was evaluated. The sodium oxychlorosene used in theseevaluations was sold under the name Clorpactin WCS-90 (manufactured byGuardian Labs and sold by Cardinal Health). Sodium oxychlorosene hasbeen used to treat urinary tract infections and in abdominal and plasticsurgery.

[0202] Results for pretreatment of the bladder surface with sodiumoxychlorosene are shown in FIGS. 8-18. FIGS. 8A-8N are photographsshowing seven murine bladders after pretreatment with a 0.2%oxychlorosene solution for 5 minutes followed by infection with Ad-LacZ.FIGS. 8A and 8B show the outside and luminal surfaces, respectively, ofthe first bladder, FIGS. 8C and 8D show the outside and luminalsurfaces, respectively, of the second bladder, FIGS. 8E and 8F show theoutside and luminal surfaces, respectively, of the third bladder, FIGS.8G and 8H show the outside and luminal surfaces, respectively, of thefourth bladder, FIGS. 8I and 8J show the outside and luminal surfaces,respectively, of the fifth bladder, FIGS. 8K and 8L show the outside andluminal surfaces, respectively, of the sixth bladder, and FIGS. 8M and8N show the outside and luminal surfaces, respectively, of the seventhbladder.

[0203] FIGS. 9A-9N are photographs showing seven murine bladders afterpretreatment with a 0.2% oxychlorosene solution for 15 minutes followedby infection with Ad-LacZ. FIGS. 9A and 9B show the outside and luminalsurfaces, respectively, of the first bladder, FIGS. 9C and 9D show theoutside and luminal surfaces, respectively, of the second bladder, FIGS.9E and 9F show the outside and luminal surfaces, respectively, of thethird bladder, FIGS. 9G and 9H show the outside and luminal surfaces,respectively, of the fourth bladder, FIGS. 9I and 9J show the outsideand luminal surfaces, respectively, of the fifth bladder, FIGS. 9K and9L show the outside and luminal surfaces, respectively, of the sixthbladder, and FIGS. 9M and 9N show the outside and luminal surfaces,respectively, of the seventh bladder.

[0204]FIGS. 10A and 10B are photographs showing the cross section of themurine bladders of FIGS. 8C and 8I, respectively. FIGS. 11A and 11B arephotographs showing the cross section of the murine bladders of FIGS. 9Cand 91, respectively.

[0205] FIGS. 12A-12F are photographs showing the cross section of threemurine bladders after pretreatment with a 0.2% oxychlorosene solutionfor 5 minutes followed by infection with Ad-LacZ. FIGS. 12A and 12B arephotographs showing the cross-section of the first murine bladder, FIGS.12C and 12D are photographs showing the cross-section of the secondmurine bladder, and FIGS. 12E and 12F are photographs showing thecross-section of the third murine bladder. FIGS. 12A, 12C and 12E weretaken at 40× and FIGS. 12B, 12D and 12F were taken at 100×magnification.

[0206] FIGS. 13A-13F are photographs showing the cross section of threemurine bladders after pretreatment with a 0.2% oxychlorosene solutionfor 15 minutes followed by infection with Ad-LacZ. FIGS. 13A and 13B arephotographs showing the cross-section of the first murine bladder, FIGS.13C and 13D are photographs showing the cross-section of the secondmurine bladder, and FIGS. 13E and 13F are photographs showing thecross-section of the third murine bladder. FIGS. 13A, 13C and 13E weretaken at 40× and FIGS. 13B, 13D and 13F were taken at 100×magnification.

[0207]FIG. 14A is a photograph showing the luminal surface of a murinebladder after pretreatment with a 0.1% oxychlorosene solution followedby infection with Ad-LacZ. FIGS. 14B and 14C are photographs showing thecross section of the murine bladder of FIG. 14A. FIG. 14B was taken at40× and FIG. 14C was taken at 100× magnification;

[0208]FIG. 15A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.2% oxychlorosene solutionfollowed by infection with Ad-LacZ. FIGS. 15B and 15C are photographsshowing the cross section of the murine bladder of FIG. 15A. FIG. 15Bwas taken at 40× and FIG. 15C was taken at 100× magnification.

[0209]FIG. 16A is a photograph showing the luminal surface of a secondmurine bladder after pretreatment with a 0.2% oxychlorosene solutionfollowed by infection with Ad-LacZ. FIGS. 16B and 16C are photographsshowing the cross section of the murine bladder of FIG. 16A. FIG. 16Bwas taken at 40× and FIG. 16C was taken at 100× magnification.

[0210]FIG. 17A is a photograph showing the luminal surface of a firstmurine bladder after pretreatment with a 0.4% oxychlorosene solutionfollowed by infection with Ad-LacZ. FIGS. 17B and 17C are photographsshowing the cross section of the murine bladder of FIG. 17A. FIG. 17Bwas taken at 40× and FIG. 17C was taken at 100× magnification.

[0211]FIG. 18A is a photograph showing the luminal surface of a secondmurine bladder after pretreatment with a 0.4% oxychlorosene solutionfollowed by infection with Ad-LacZ. FIGS. 18B and 18C are photographsshowing the cross section of the murine bladder of FIG. 18A. FIG. 18Bwas taken at 40× and FIG. 18C was taken at 100× magnification.

[0212] Polymers with Alternating Hydrophilic and Lipophilic Units

[0213] Polymeric compounds comprising repeating sequences of alternatingor identical monomers were also tested. One such compound tested wasPoloxamer 407 (Pluronic 127) having a structure represented by thefollowing formula:

[0214] Poloxamers polymers come in a wide range of HLB values. Both ofthe compounds tested, however, had only a minimal effect on thetransduction of adenovirus. While not wishing to be bound by theory, itis believed that compounds having separated, longer hydrophilic andlipophilic chains are more effective at enhancing transduction of thebladder epithelium.

[0215] Additional Transduction Enhancing Compounds

[0216] Additional compounds can also be used as transduction enhancingagents according to the invention.

[0217] These compounds include ω-undecylenyl-β-D-maltopyranoside, whichhas a structure represented by:

[0218] Sugar based thiolic compounds such asalkyl-β-D-thioglucopyranosides having a general structure representedby:

[0219] may also be employed.

[0220] Additionally, alkyl-β-D-thiomaltopyranosides having a generalstructure represented by:

[0221] may also be used as transduction enhancing compounds according tothe invention.

[0222] Further, compounds having a positive charge such as

[0223] can also be used.

[0224] Additionally, compounds wherein the lipophilic and hydrophilicparts are connected via a carboxylic bond can also be employed. Anexemplary compound of this type is6-O-methyl-n-heptylcarboxyl-α-D-glucopyranoside:

[0225] Sugar based compounds having alkyl groups with side groups orother modifications may also be used. Exemplary compounds of this typeinclude 2-propyl-1-pentyl-β-D-maltopyranoside having a structurerepresented by:

[0226] Sarcosine compounds may also be used as transduction enhancingagents according to the invention. Exemplary sarcosine compounds includesodium alkyl sarcosine having a structure represented by:

[0227] Various substituted sugars can also be used as transductionenhancing compounds. An exemplary substituted sugar which can be used asa transduction enhancing compound is a sucrose mono alkyl ester having achemical structure represented by:

[0228] Exemplary compounds of this type include compounds wherein n=10(i.e., sucrose monolaurate).

[0229] Also according to the present invention, methods of treating theluminal surface of the bladder are provided. According to a preferredembodiment of the invention, the bladder is treated by instillationusing bladder catheterization. According to this embodiment, any urinein the bladder is first removed and the bladder is optionally washedwith a buffer (e.g., PBS). A composition comprising the transductionenhancing agent is then applied to the luminal surface of the bladder(e.g., by instillation). The transduction enhancing solution may beincubated for some specified time or drained immediately. Multipletreatments with the composition comprising the transduction enhancingagent can be performed. After treatment with the transduction enhancingagent, the luminal surface of the bladder may be washed with a buffer(e.g., PBS). A solution comprising the adenovirus can then be introducedinto the bladder (e.g., by instillation). The solution comprising theadenovirus can be removed immediately or, alternatively, the solutioncan be allowed to incubate for a certain amount of time. After treatmentwith the adenovirus, the bladder surface can again be washed with abuffer solution (e.g., PBS). According to a preferred embodiment of theinvention, about 50 to about 500 ml of the transduction enhancingcomposition is delivered to the bladder by instillation for eachtreatment.

[0230] Alternatively, a composition comprising the transductionenhancing agent and the adenovirus can be used to treat the luminalbladder surface. According to this embodiment of the invention, anyurine in the bladder is first removed and the bladder is then optionallywashed with a buffer (e.g., PBS). A composition comprising thetransduction enhancing agent and the adenovirus is then applied to theluminal surface of the bladder. The solution may be incubated for somespecified time or drained immediately. After treatment, the luminalsurface of the bladder may again be washed with a buffer (e.g., PBS).

[0231] Although phosphate buffered saline (PBS) is the preferred buffer,any other pharmaceutical buffer can be used according to the invention.Exemplary buffers include sodium phosphate/sodium sulfate, Tris buffer,glycine buffer, sterile water and other buffers known in the art,including those described by Good, et al., Biochemistry 5, 467 (1966).The pH of the buffer can be in the range of 6.4 to 8.4, preferably 7 to7.5, and most preferably 7.2 to 7.4.

[0232] The composition comprising the transduction enhancing agentaccording to the invention preferably also comprises an oxidizing agent.Exemplary oxidizing agents include, but are not limited to, chloritecompounds, hypochlorous acid, hydrogen peroxide, and peroxyacetic acid.According to a preferred embodiment of the invention, any of the singlecompound transduction enhancing agents can be combined with an oxidizingagent and used as a transduction enhancing agent.

[0233] As set forth above, the viral gene therapy vehicle can be anoncolytic virus, for example an oncolytic adenovirus exemplified hereinby CG8840. The adenovirus composition can further comprise achemotherapeutic agent such as Docetaxel. The adenovirus compositionpreferably comprises from about 1×10¹¹ to about 1×10¹⁴ viral particles.

[0234] While the foregoing specification teaches the principles of thepresent invention, with examples provided for the purpose ofillustration, it will be appreciated by one skilled in the art fromreading this disclosure that various changes in form and detail can bemade without departing from the true scope of the invention.

[0235] All publications cited herein are hereby incorporated byreference in their entirety.

What is claimed is:
 1. A method for treating cancer of the bladdercomprising: contacting the luminal surface of the bladder with apretreatment composition comprising a transduction enhancing agent; andsubsequently contacting the luminal surface of the bladder with acomposition comprising an oncolytic virus; wherein the transductionenhancing agent is a mono-, di-, or poly-saccharide having a lipophilicsubstituent.
 2. The method of claim 1, wherein the transductionenhancing agent is a di-saccharide having a lipophilic substituent. 3.The method of claim 1, wherein the transduction enhancing agent is adi-saccharide having a lipophilic substituent and wherein thedi-saccharide is selected from the group consisting of sucrose, lactose,maltose, isomaltose, trehalose and cellobiose.
 4. The method of claim 1,wherein the transduction enhancing agent has the following generalformula (I) or the following general formula (II):

wherein X is a sulfur or oxygen atom, each R² is independently hydrogenor a moiety represented by:

and R¹ represents an alkyl or alkenyl group.
 5. The method of claim 1,wherein the transduction enhancing agent has the chemical formula:

wherein n is a positive integer.
 6. The method of claim 5, wherein n is11 or greater.
 7. The method of claim 5, wherein n is
 11. 8. The methodof claim 7, wherein the pretreating composition comprises about 0.02 toabout 0.5% by weight of the transduction enhancing agent.
 9. The methodof claim 1, wherein the transduction enhancing agent has the chemicalformula:

wherein n is a positive integer.
 10. The method of claim 9, wherein n is6 or greater.
 11. The method of claim 9, wherein n is
 6. 12. The methodof claim 11, wherein the pretreatment composition comprises about 0.1%by weight of the transduction enhancing agent.
 13. The method of claim1, wherein the oncolytic virus is an oncolytic adenovirus.
 14. Themethod of claim 13, wherein the oncolytic adenovirus is CG8840.
 15. Themethod of claim 13, wherein the oncolytic virus composition furthercomprises a chemotherapeutic agent.
 16. The method of claim 15, whereinthe chemotherapeutic agent is docetaxel.
 17. The method of claim 1,wherein contacting the luminal surface of the bladder with a compositioncomprising an oncolytic virus comprises delivering about 50 to about 500ml of the oncolytic virus composition to the bladder by instillation.18. The method of claim 1, wherein the oncolytic virus compositioncomprises from about 1×10¹¹ to about 1×10¹⁴ viral particles.
 19. Themethod of claim 1, wherein contacting the luminal surface of the bladderwith a pretreatment composition comprises delivering the pretreatmentcomposition to the bladder by instillation.
 20. The method of claim 1,further comprising washing the luminal surface of the bladder aftercontact with the pretreatment composition and before contact with theoncolytic virus composition.
 21. The method of claim 1, wherein thepretreatment composition is contacted with the luminal surface of thebladder for about 5 minutes.
 22. The method of claim 1, wherein thepretreatment composition further comprises an oxidizing agent.
 23. Themethod of claim 22, wherein the oxidizing agent is selected from thegroup consisting of hypochlorous acid, hydrogen peroxide, andperoxyacetic acid.
 24. The method of claim 1, wherein the lipophilicsubstituent comprises an alkane group.
 25. The method of claim 1,wherein the lipophilic substituent is an alkanoic acid residue.
 26. Themethod of claim 1, wherein the transduction enhancing agent has thechemical formula:

where R¹ represents an alkyl or alkenyl group.
 27. The method of claim26, wherein R¹ is represented by:


28. A method for treating cancer of the bladder comprising: contactingthe luminal surface of the bladder with a pretreatment compositioncomprising about 0.01 to about 0.2% by weight sodium oxychlorosene; andsubsequently contacting the luminal surface of the bladder with acomposition comprising an oncolytic virus.
 29. The method of claim 28,wherein the pretreatment composition comprises from about 0.01 to about0.1% by weight sodium oxychlorosene.
 30. The method of claim 28, whereinthe oncolytic virus is an oncolytic adenovirus.
 31. The method of claim30, wherein the oncolytic adenovirus is CG8840.
 32. The method of claim30, wherein the oncolytic virus composition further comprises achemotherapeutic agent.
 33. The method of claim 32, wherein thechemotherapeutic agent is docetaxel.
 34. The method of claim 28, whereinthe oncolytic virus composition comprises from about 1×10¹¹ to about1×10¹⁴ viral particles.
 35. The method of claim 28, further comprisingwashing the luminal surface of the bladder after contact with thepretreatment composition and before contact with the oncolytic viruscomposition.
 36. A method of treating cancer of the bladder comprising:contacting the luminal surface of the bladder with a pretreatmentcomposition comprising a transduction enhancing agent having a structurerepresented by the chemical formula:

wherein x and y are positive integers; and subsequently contacting theluminal surface of the bladder with a composition comprising anoncolytic virus.
 37. The method of claim 36, wherein x is 6 and y is8-10.
 38. The method of claim 37, wherein the pretreatment compositioncomprises about 0.02 to about 0.5 wt. % of the transduction enhancingagent.
 39. The method of claim 36, wherein the oncolytic virus is anoncolytic adenovirus.
 40. The method of claim 39, wherein the oncolyticadenovirus is CG8840.
 41. The method of claim 39, wherein the oncolyticvirus composition further comprises a chemotherapeutic agent.
 42. Themethod of claim 41, wherein the chemotherapeutic agent is docetaxel. 43.The method of claim 36, wherein the pretreatment composition furthercomprises an oxidizing agent.
 44. The method of claim 43, wherein theoxidizing agent is selected from the group consisting of hypochlorousacid, hydrogen peroxide, and peroxyacetic acid.
 45. A method of treatingcancer of the bladder comprising: contacting the luminal surface of thebladder with a pretreatment composition comprising a transductionenhancing agent having a structure represented by the following generalformula (I) or the following general formula (II):

wherein x is a positive integer; and subsequently contacting the luminalsurface of the bladder with a composition comprising an oncolytic virus.46. The method of claim 45, wherein x is
 11. 47. The method of claim 46,wherein the transduction enhancing agent has a structure represented bythe general formula (I).
 48. The method of claim 47, wherein thepretreatment composition comprises about 0.1 wt. % of the transductionenhancing agent.
 49. The method of claim 46, wherein the transductionenhancing agent has a structure represented by the general formula (II).50. The method of claim 49, wherein the pretreatment compositioncomprises about 0.2 wt. % of the transduction enhancing agent.
 51. Themethod of claim 45, wherein the oncolytic virus is an oncolyticadenovirus.
 52. The method of claim 51, wherein the oncolytic adenovirusis CG8840.
 53. The method of claim 51, wherein the oncolytic viruscomposition further comprises a chemotherapeutic agent.
 54. The methodof claim 53, wherein the chemotherapeutic agent is docetaxel.
 55. Themethod of claim 45, wherein the pretreatment composition furthercomprises an oxidizing agent.
 56. The method of claim 55, wherein theoxidizing agent is selected from the group consisting of hypochlorousacid, hydrogen peroxide, and peroxyacetic acid.
 57. A compositioncomprising: a transduction enhancing agent; and an oncolytic virus;wherein the transduction enhancing agent is a mono-, di-, orpoly-saccharide having a lipophilic substituent.
 58. The composition ofclaim 57, wherein the transduction enhancing agent is a compound havingthe following general formula (I) or the following general formula (II):

wherein X is a sulfur or oxygen atom, each R² is independently hydrogenor a moiety represented by:

and R¹ represents an alkyl or alkenyl group.
 59. The composition ofclaim 57, wherein the oncolytic virus is an oncolytic adenovirus. 60.The composition of claim 57, wherein the oncolytic adenovirus is CG8840.61. The composition of claim 57, further comprising a chemotherapeuticagent.
 62. The composition of claim 61, wherein the chemotherapeuticagent is docetaxel.
 63. A method for treating cancer of the bladdercomprising contacting the luminal surface of the bladder with thecomposition of claim
 57. 64. A composition comprising sodiumoxychlorosene and an oncolytic virus.
 65. The composition of claim 64,wherein the oncolytic virus is an oncolytic adenovirus.
 66. Thecomposition of claim 64, wherein the oncolytic adenovirus is CG8840. 67.The composition of claim 64, further comprising a chemotherapeuticagent.
 68. The composition of claim 67, wherein the chemotherapeuticagent is docetaxel.
 69. The composition of claim 64, wherein thecomposition comprises about 0.01 to about 0.4% by weight sodiumoxychlorosene
 70. The composition of claim 64, wherein the compositioncomprises about 0.01 to about 0.2% by weight sodium oxychlorosene
 71. Amethod for treating cancer of the bladder comprising contacting theluminal surface of the bladder with the composition of claim 64.